Fig 6.

PB1-F2 expression is differentially regulated by the PB1-F2 ORF and 3′ PB1 regions. (A) Diagrams of chimeric PB1 gene plasmids constructed to identify sequence requirements for differential PB1-F2 expression. The PB1 gene was divided into three sections consisting of the 5′ region upstream of PB1-F2, the PB1-F2 ORF, and the 3′ region downstream of PB1-F2 in the 3×FLAG-tagged PB1-F2 plasmids created for Fig. 5. All possible combinations of the PB1 gene using these regions from OH07, Mx09, or PR8 were created. (B) MDCK cells were transfected with the indicated plasmids, and at 24 h posttransfection, the cells were immunostained with antibodies against PB1 and 3×FLAG, followed by Alexa Fluor-conjugated secondary antibodies. The percentage of PB1-expressing cells also expressing detectable amounts of PB1-F2/3×FLAG was determined and plotted on a graph. Statistically significant groups (P < 0.01) as determined by ANOVA are indicated with a line and two, three, four, or five asterisks. The error bars indicate standard deviations.